Prostate Specific Antigen

PSA was first purified from prostatic tissue and was shown to be prostate specific in 1979 [26]. One year later, it was found to be present in serum of patients with prostate cancer [27, 28]. Although PSA was later shown to be produced also in other tissues, it is in practice a specific marker for prostate cancer and prostatic diseases.

3.1. Biochemistry and Structure of PSA

PSA (also called hK3) is a 33-kDa serine protease with chymotrypsin-like enzymatic activity [29-31]. It is encoded by a gene clustered in chromosomal region 19q13.4, together with hK2, tissue kallikrein (hK1), and 12 other characterized structurally related human kallikrein genes [30-33]. The genes of the human kallikrein family are numbered KLK1-15, and the corresponding proteins named hK1-15. All members of the kallikrein family have five exons and display considerable (40-79%) sequence similarities at the DNA and amino acid levels [29,30, 32-34]. The best-known kallikreins are hK1, hK2, and PSA.

PSA and hK2 are the most closely related, with 79% identity in amino acid sequence, while hK1 displays 62% identity with PSA [34].

PSA is a single-chain glycoprotein. The structure deduced from the cDNA sequence shows that PSA is synthesized as a 261 amino acid preproenzyme, comprising a 17-amino acid signal peptide, followed by a 7-amino acid propeptide and a 237-amino acid mature, enzymatically active protein [35, 36]. The signal peptide is removed during synthesis. The secreted proenzyme, called proPSA, is enzymatically inactive and can be activated into the mature form by proteolytic cleavage with trypsin, hK2, hK4, and prostin [37-41]. Trypsin is the most efficient activator, followed by hK4, prostin, and hK2 [37, 40-42]. PSA also contains a carbohydrate chain linked to Asn-45 (numbering according to active PSA). The average molecular weight of mature PSA determined by mass spectrometry is 28430 [43], and the relative molecular mass determined by SDS-PAGE and gel filtration is about 33 kDa [43-45].

PSA contains a catalytic triad that is characteristic to serine proteases. Residues His-41, Asp-96, and Ser-189 are located in typical sites of the active pocket [46, 47]. Ser-183, which is located at the bottom of the active site, is crucial for the specificity of PSA [48]. The enzymatic activity of PSA is chymotrypsin-like, but very restricted, and it preferentially hydrolyzes pep-tide bonds at the carboxy-terminus of the hydrophobic residues tyrosine and leucine [49, 50]. Several residues surrounding these preferred P1 residues play an important role in determining the substrate specificity [50-52].

3.2. Expression of PSA 3.2.1. Tissue Expression of PSA

PSA is secreted by normal prostatic epithelial cells, benign prostatic hyperplasia, and prostate cancer cells, and its expression is stimulated by androgens through androgen receptor mediated transcriptional activation [26, 44, 53-55]. In LNCaP cells, which are the most widely used model for studying PSA expression, dihydrotestosterone is the most potent inducer of PSA synthesis [56]. Several growth factors and other factors have also been suggested to affect PSA expression [57, 58].

In the prostate, the secretion of PSA is directed into the prostatic ducts, and PSA occurs at very high concentrations, 0.5-2 mg/ml, in human seminal fluid [45]. In seminal fluid, most of the PSA occurs in an intact, enzymatically active form, while 35-40% is internally cleaved and inactive (nicked PSA) [49, 59]. ProPSA has not been detected in seminal fluid [60].

PSA is quite organ specific, but it has been shown to be weakly expressed in some other human tissues, for example, in the periurethral glands, the anal gland, and breast tissue of males and females [61-64]. Low expression of PSA

has also been detected in the gastrointestinal tract [65]. PSA can occasionally be found in other cancers such as adrenal, kidney, lung, and colon cancers, and it has also been used as a prognostic marker for breast cancer [66-69]. With ultrasensitive immunoassays, PSA can be detected at very low concentrations in female serum and other body fluids, including amniotic fluid, breast fluid, cyst fluid, and nipple aspirate fluid [70-72].

The expression of PSA is higher in benign prostatic tissue than in cancer [73], but normal epithelial cells of the prostate secrete PSA into the glandular ducts, and it reaches circulation only by leaking into interstitial space and then diffusing into circulation [74]. Thus, the serum concentrations are normally about 1 million-fold lower than those in seminal fluid. In prostate cancer, however, the tissue architecture of the prostate is deranged, and when the tumor loses connection with the prostatic ducts PSA is released directly into the interstitial space and circulation [74, 75]. This explains why a prostate cancer produces about 30-fold higher serum concentrations of PSA per gram tissue than the normal prostate [2]. The PSA released from prostate cancer is thought to be more active than the PSA leaking out from benign prostatic tissue. This probably explains why the proportion of PSA occurring in complex with ACT is increased in serum from prostate cancer patients [12, 75].

3.2.2. PSA-Producing Cell Lines

Several of the prostate cancer cell lines used in research produce PSA [76, 77]. Although the amount and activity of the PSA produced by these cell lines are much lower than in normal prostate tissue or primary human prostate cancers [78], these cell lines are valuable for studying the regulation and function of PSA. The LNCaP cell line, isolated from a needle biopsy of a lymph node metastatic lesion, is the most widely used PSA-producing prostate cancer cell line [76, 77, 79]. When grown in a medium containing fetal bovine serum, LNCaP cells mainly secrete PSA as the inactive proenzyme [42, 80]. The amount of enzymatically active PSA increases when the cells are grown in a medium without serum [80]. LNCaP cells express androgen and estrogen receptors, but the androgen receptor contains a mutation that enables the receptor to also bind some other steroids [81]. In addition to the parental line, there are also over 50 LNCaP cell sublines with variable steroid responses.

3.3. Isoforms of PSA from Seminal Fluid

Most of the PSA purified from seminal fluid is in an intact, enzymatically active form, while 35-40% is internally cleaved and inactive (also called nicked PSA) [29, 49, 59, 60]. Purified PSA can be further subfractionated by reverse phase [29] and anion exchange chromatography [59]. By using anion exchange chromatography, purified PSA from seminal fluid can be separated into five isoforms (A, B, C, D, and E). Isoforms PSA-A and -B are intact enzymatically active forms, which differ in glycosylation, while PSA-C, -D, and -E are internally cleaved forms [59]. The internal cleavage sites in the peptide backbone are at Arg-85, Lys-145, and Lys-182. The cleavages disrupt the conformation of PSA, causing loss of catalytic activity [29, 49, 59]. The fragments in cleaved PSA molecules are held together by disulfide bonds, but they can be separated in SDS-PAGE under reducing conditions [59].

Intact PSA forms complexes with A2M, pregnancy zone protein, ACT, protein C inhibitor, and API when incubated with these protease inhibitors in vitro [14, 49, 59, 82]. When added to human serum, PSA preferentially forms complexes with A2M and ACT [83], but it also forms a complex with API [14, 84]. The complex between PSA and protein C inhibitor has only been found in human seminal plasma [82, 85]. The complexes of PSA-ACT and PSA-API dissociate gradually during storage in solution. The released PSA is enzymatically active and can form a complex with A2M [14, 83].

Nicked isoforms of PSA (PSA-C, -D, and -E) are very inefficient in forming complexes with ACT and API as compared to intact PSA, but 40-80% of these nicked forms react with A2M, though the rate is slow [59, 83]. The binding of PSA with A2M requires enzymatic cleavage of the bait region of A2M, resulting in a conformational change and encapsulation of the protease by A2M. Thus, the ability of nicked PSA to form a complex with A2M is surprising.

3.4. Biological Functions of PSA

The main biological function of PSA is liquefaction of the seminal gel formed after ejaculation by proteolytic cleavage of semenogelin I and II that are physiological substrates of PSA and major proteins in seminal fluid [86]. Some studies have suggested that PSA plays a role both in inhibition and promotion of prostate cancer invasion and metastasis. PSA may inhibit cell growth and angiogenesis by generating angiostatin-like fragments from plas-minogen [87-89], and it may also induce apoptosis [90]. Other experiments suggest that PSA activates the urokinase-type plasminogen activator that is thought to be involved in tumor invasion and metastasis [91]. PSA might also affect tumor spread by proteolytic modulation of cell adhesion receptors [92]. Furthermore, PSA has been found to cleave insulin-like growth factor binding protein-3 and -4 (IGFBP-3 and -4), causing local release of active insulin-like growth factor-I (IGF-I) that could enhance tumor growth [93, 94]. However, prostate cancer patients with high concentrations of PSA in serum do not have an increased proportion of cleaved IGFBP-3

[95]. PSA also cleaves fibronectin and laminin [96], and activates latent TGF-,3 [92]. By these latter mechanisms PSA may mediate progression of prostate cancer. It is not clear, which function of PSA is most important or even physiologically relevant, but based on the association between low-tissue concentrations of PSA and adverse prognosis [97], it is conceivable that PsA prevents rather than promotes progression of prostate cancer.

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